The present invention relates to the use of continuous mammalian cell lines that support the growth and productive infection of Marek""s Disease Virus (MDV) at high titers. The present invention relates to a cell line that can be used as a substrate to efficiently propagate large quantities of Marek""s Disease Virus, in particular, for vaccine production. The present invention relates to recombinant Marek""s Disease Virus vectors and cell lines to package said vectors and recombinant Marek""s disease viruses which may be used as vaccines. Marek""s Disease Virus vectors and vaccines may be used to protect avians from infection with Marek""s Disease Virus and against disease resulting from infection.
Marek""s Disease (MD) is an acutely oncogenic disease of chickens, which causes lymphomas, visceral tumors, nerve lesions and immunosuppression. The disease is global and ubiquitous in distribution. The etiologic agent is herpesvirus, Marek""s Disease Virus. Marek""s Disease has been a primary cause of deaths and condemnations in broiler flocks [Calnek, B. W. and Witter, R. L., Diseases of Poultry, 9th edition, Iowa State Press, Ames, Iowa, pp. 342-385 (1991)].
There are three serotypes of Marek""s Disease Virus. Serotype 1 includes all pathogenic strains and their attenuated derivatives. Serotype 2 consists of naturally avirulent chicken viruses, while serotype 3, also known as Herpesvirus of Turkeys (HVT), includes avirulent turkey viruses that are capable of replication in chickens. The three serotypes are partially cross-protective, but can be distinguished using monoclonal antibodies [Silva, R. F. and Lee, L. F., Virol. vol. 36. pp. 307-320 (1984)] and other known methods.
Marek""s Disease Virus appears to be less recombinogenic than other herpes viruses, and the cell-associated nature of the virus makes plaque purification of recombinants away from parental virus problematic. Nevertheless, recombinant MD viruses have been generated from serotype 1 [Sonoda, K. et al., Vaccine V. 14, pp. 277-284 (1996); Parcells, M. S., et al., J. Virol., V. 69. pp. 7888-7898 (1995); Parcells M. S., et al., Virus Genes, V. 9, pp. 5-13, (1994); Parcel M. S., et al., J. Virol., V. 68, pp. 8239-8253 (1994); Sakaguchi, M., Vaccine V. 12, pp. 953-957 (1994); Reddy, S. K., et al., Vaccine V. 14, pp. 469-477 (1996)], serotype 2 [Marshall, D. R., et al., Virol. V. 195, pp. 638-648 (1993); Silva, R. F., 14th International Herpesvirus Workshop (Abstract) (1989)]; and serotype 3 [Reddy, S. K., et al., Vaccine V. 14, pp. 469-477 (1996); PCT Parent Application WO 95/29248 (1995); Silva, R. F., 14th International Herpesvirus Workshop (Abstract) (1989); Darteil, R., et al., Virol. V. 211, pp. 481-490 (1995); U.S. Pat. No. 5,187,087 issued in 1995; Zelnik, V., et al., J. Gen. Virol. V. 76, pp. 2903-2907 (1995); PCT Patent Application WO 93/25665, published (1993); Ross, L. J. N., et al., J. Gen. Virol., V. 74, pp. 371-377 (1993), Sondermeijer, P. J. A., et al., Vaccine V. 11, pp. 349-358 (1993); European Patent No. 431,668 B1, published (1995); Morgan, R. W., et al., Avian Dis., V. 36, pp. 858-870 (1992); Bandyopadhyay, P. K., et al., 13th International Herpesvirus Workshop 323 (Abstract) (1988)]. In all of the above mentioned references, the viruses were replication competent and were produced in primary avian cells.
Commercially available Marek""s Disease Virus vaccines, with the exception of some monovalent HVT formulations, consist largely of live Marek""s Disease Virus-infected primary chicken embryo fibroblast (CEF) cells. A significant problem associated with using whole live Marek""s Disease Virus-infected primary chicken cells to grow Marek""s Disease Virus for use in vaccines is that the CEF cells must be stored at liquid nitrogen temperatures and administered by injection in order to be effective. Whole live cell vaccines have been previously necessary since the three Marek""s Disease Virus serotypes are strongly cell-associated in cell culture and in most tissues of an infected bird. Spread of infection within birds can be achieved by direct cell to cell contact, with little or no cell-free virus being released. Infectious virions are produced only in the feather follicle epithelium (FFE), and are responsible for bird-to-bird transmission [Calnek, B. W., et al., Avian Dis., V. 14, pp. 219-233 (1970); Witter, R. L., et al., J. Natl. Cancer Inst., V. 49, pp. 1121-1130 (1972); Edison, C. S., et al., J. Natl. Cancer Inst., V. 47, pp. 113-120 (1971)].
Commercial cell-free Marek""s Disease Virus vaccines can be made by cell culture. However, the production of cell-free Marek""s Disease Virus vaccines has been thus far been limited to vaccines produced using only serotype 3 Marek""s Disease Virus. This is because only serotype 3 Marek""s Disease Virus makes free virions in sufficient quantities for production of Marek""s Disease Virus vaccines. It has been suggested that lack of expression of the viral glycoprotein D (gD) gene may be involved in limiting release of cell-free virions [PCT Patent Application WO 95/29248 published (1995); Tan, X. and Velicer, L. F., 18th International Herpesvirus Workshop A, 145 (Abstract) (1993)].
In addition to CEF, other primary avian cells have been used to grow Marek""s Disease Virus, including chicken embryo kidney (CEK) and duck embryo fibroblast (DEF). Only two continuous avian cell lines have been described as being capable of growing certain Marek""s Disease Virus serdtypes. Serotype 3 Marek""s Disease Virus (Marek""s Disease Virus-3) has been described as growing on a chemically transformed quail cell line designated QT35 [Nikura, M., Nanta, T. et al., J. Vet. Med. Sci., V. 53, pp. 439-446 (1991)]. A chemically transformed CEF cell line designated CHCC-OU2 [Ogura, H. and Fujiwara, T., Acta Med. Okayama, V. 41, pp. 141-143 (1987)] has been described as supporting the growth of Marek""s Disease Virus-1 [Abujoub, A. and Coussens, P. M., Virol., V. 214, pp. 541-549 (1985)].
Other known processes for producing Marek""s Disease Virus include the use of tumorigenic or oncogenic cell lines. Marek""s Disease Virus-transformed lymphoblastoid cell lines [Nazerian, K. Avian Pathol. V. 16, pp. 527-544 (1987)] are derived from lymphoid tumors in chickens infected with oncogenic Marek""s Disease Virus-1. The viral genome is maintained in a latent or semi-latent state in these cells, such that transmission of infection to co-cultivated CEF cells or DEF cells occurs at a low frequency, if at all. In addition, these lymphoblastoid lines are refractory to superinfection with other Marek""s Disease Virus viruses. Furthermore, Marek""s Disease Virus-transformed lymphoblastoid cell lines have not demonstrated utility in the production of nonrecombinant (conventional) Marek""s Disease Virus vaccines or in the preparation of recombinant Marek""s Disease Virus viruses or genetically altered Marek""s Disease Virus viruses or vectors.
Similarly, lymphoblastoid cell lines [Nazerian, K., Avian Pathol., V. 16, pp. 527-544 (1987)] derived from oncogenic avian retroviruses (avian leukosis virus and reticuloendotheliosis virus) are not useful for the production of commercial Marek""s Disease Virus vaccines or for the generation of recombinant Marek""s Disease Virus, due to the shedding of retroviruses, the poor growth characteristics of lymphoblastoid cells, and the low level of productive Marek""s Disease Virus infection.
Mammalian primary cells and mammalian cell lines have never been described as suitable substrates for the growth of any serotype of Marek""s Disease Virus, nor has any mammal ever been demonstrated to be productively infected with Marek""s Disease Virus.